Hydrogen in Pd/Nb multilayers.
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Journal Article↗
Biomedical subjects
G Schmitz
Publications and source records attributed to G Schmitz.
Genetic and biochemical evidence that CESD and Wolman disease are distinguished by residual lysosomal acid lipase activity.
Cholesteryl ester storage disease (CESD) and Wolman disease are both autosomal recessive disorders associated with reduced activity and genetic defects of lysosomal acid lipase (LAL). We provide evidence that the strikingly more severe course of Wolman disease is caused by genetic defects of LAL that leave no residual enzyme activity. In a CESD patient, a G --> A mutation at position -1 of the exon 8 splice donor site results in skipping of exon 8 in 97% of the LAL hnRNA originating from this allele, while 3% are spliced correctly, resulting in full-length LAL enzyme. The mutant LAL mRNA codes for a protein lacking amino acids 254 to 277. On the other allele, a G --> T mutation leads to a premature stop codon at Gly245, resulting in inactive LAL enzyme. In addition, the previously identified Leu179 --> Pro mutation is present on this allele, and the LAL mRNA is rendered unstable by the premature stop codon. Analysis of two children with Wolman disease showed that both were homozygous for a G --> A mutation at position +1 of the same splice donor site as for the CESD patient, leading to skipping of exon 8. In contrast to the CESD patient, no correctly spliced mRNA was detectable. We have also expressed a wildtype LAL cDNA and the mutant LAL cDNA from one Wolman patient in Sf9 and H5 insect cells. We demonstrate that the LAL enzyme generated from the wildtype LAL cDNA was active in homogenates from Sf9 and H5 cells, while the enzyme with the internal deletion of 24 amino acids originating from the LAL cDNA of the Wolman patient was not. The combined data provide evidence that the only functionally relevant genetic difference between the Wolman patients and the CESD patient is that the splice defect in Wolman, which affects one of the invariable nucleotides of the splice consensus sequences (position +1), does not permit any correct splicing, whereas the defect observed in CESD (position -1) allows some correct splicing (3% of total LAL mRNA) and therefore the synthesis of functional enzyme.
Alternative Splicing↗
Characterization of a plastid-specific HSP90 homologue: identification of a cDNA sequence, phylogenetic descendence and analysis of its mRNA and protein expression.
The isolation of cDNAs is described which encode the complete sequence of a precursor protein for a HSP90 homologue consisting of an N-terminal transit peptide of 5850 Da and a mature protein (cpHSP82) of 82 260 Da, located in the plastids of rye leaves (Secale cereale). Hybridization analysis indicated the presence of a single gene in the DNA of rye and a transcript size of 2.8 kb. A phylogenetic tree constructed on the basis of sequence comparisons for HSP90 homologues from different species and compartments indicated that the plastidic HSP82 from rye was more closely related to an eubacterial protein than to HSP90 homologues of the cytosol or ER from both plants and animals. The results suggest that during chloroplast evolution the gene for cpHSP82 was transferred to the nucleus from a prokaryotic endosymbiont. Immunoblots with specific antibodies and Percoll gradient-purified organelles confirmed the location of cpHSP82 in chloroplasts or non-green plastids. In green rye leaves cpHSP82 was constitutively expressed and equally distributed among tissues of different age. The expression of cpHSP82 was enhanced within 2 h by exposure to 42 degrees C. The cpHSP82 transcript and protein were much more strongly expressed in non-green tissues, such as etiolated, 70S ribosome-deficient 32 degrees C-grown, or herbicide-bleached, than in normal green leaves. Also chromoplasts from the pericarp of tomato fruits contained high levels of a HSP90 polypeptide while a photosynthetic protein, the large subunit of ribulose-1,5-bisphosphate carboxylase was largely degraded during ripening.
Amino Acid Sequence↗
Comparison of the expression of a plastidic chaperonin 60 in different plant tissues and under photosynthetic and non-photosynthetic conditions.
A partial cDNA which codes for the beta-subunit of a plastidic chaperonin 60 (cpn60-beta) from rye (Secale cereale L.) leaves was identified and sequenced, except for 46 amino acids of the N-terminus of the mature protein and the transit sequence. This is the first cpn60-beta sequence determined for a monocotyledonous plant. Specific antibodies against cpn60-beta were affinity-purified from an antiserum raised against the total soluble protein fraction of ribosome-deficient plastids. The localization of cpn60-beta in chloroplasts or non-green plastids was confirmed by immunodetection in Percoll gradient-purified organelles. The expression and occurrence of cpn60-beta was analysed by immunoblotting with the specific antibodies and Northern hybridization. The cpn60-beta protein was constitutively expressed in various green and non-green tissues. It was evenly distributed along the major part of a rye leaf, while highest transcript levels occurred in the youngest and oldest leaf sections. The expression of the cpn60-beta protein was not enhanced by a heat-shock treatment at 42 degrees C. The cpn60-beta transcript and protein were more strongly expressed in various non-green, for instance etiolated, 70S-ribosome-deficient 32 degree C-grown, or herbicide-bleached tissues, than in green leaves of rye. A rapid increase in the cpn60-beta transcript level was also observed when green leaves were transferred from light to darkness while the protein level was not affected. The dark-induced increase in the cpn60-beta transcript was totally suppressed in the presence of 2% sucrose. Inhibitor treatments suggested that the change in cpn60-beta transcript level was not related to changes of the ATP supply of the tissue. While the large subunit of the photosynthetic protein ribulose-1,5-bisphosphate carboxylase was largely degraded during ripening of tomato fruits, high levels of cpn60-beta were detected in tomato chromoplasts and in the yellow flower petals of Narcissus. Low levels of cpn60-beta were detected in root tissue.
Amino Acid Sequence↗
Heparin effect on platelets and fibrinolysis.
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Antigens, Surface↗
Cardiopulmonary bypass leads to a preferential loss of activated platelets. A flow cytometric assay of platelet surface antigens.
OBJECTIVE: In a prospective study surface antigens associated with platelet activation, aggregation, and adhesion and the platelet volume were measured to investigate the mechanism of the platelet function defect of cardiopulmonary bypass (CPB). METHODS: Blood samples were obtained during cardiac surgery before and after heparinization, as well as during and following extracorporeal circulation. The expression of the platelet surface glycoproteins (GP) IIb-IIIa, Ib, 53, and the granule membrane protein (GMP) 140 were measured using flow cytometry in platelet-rich plasma (PRP) before and after in vitro stimulation with adenosine diphosphate. A full blood count including mean platelet volume (MPV) was taken. RESULTS: Heparinization resulted in a significant increase of GP 53 and GMP 140 and a significant decrease of GP Ib expression. During and following CPB, GP IIb-IIIa and Ib were significantly decreased. Similarly, the expression of the activation markers was reduced significantly. The mean platelet volume decreased significantly from 8.6 +/- 0.7 fl at baseline to 7.9 +/- 0.8 fl at the end of the study period. CONCLUSION: Our data suggest that heparinization induces platelet activation. We assume that a loss of larger and more activated platelets from the circulation contributes substantially to the platelet function defect of CPB.
Adult↗
Thiopentone and propofol, but not methohexitone nor midazolam, inhibit neutrophil oxidative responses to the bacterial peptide FMLP.
The effects of different anaesthetics on the neutrophil oxidative response in vitro are compared. Neutrophils were stimulated with small amounts of the bacterial peptide N-formyl-L-methionyl-L-leucyl-phenylalanine as a physiological, receptor-dependent stimulus. A new flow cytometry-based method capable of detecting the small amounts of H2O2 generated by neutrophils in a heterogeneous all-or-none response following submaximal stimulation with N-formyl-L-methionyl-L-leucyl-phenylalanine was used. Propofol and thiopentone suppressed the respiratory burst significantly (P < 0.05 and P < 0.01) in therapeutic concentrations, while midazolam and methohexitone inhibited significantly (P < 0.01) only at concentrations greater than the therapeutic range. Impairment of the neutrophil response was primarily because of a reduction in the number of neutrophils participating in the respiratory burst and not of a proportional decrease of the fluorescence of all neutrophils.
Adult↗
The effect of cysteine modification and proteinases on the major antigens (D, C, c, E and e) of the Rh blood group system.
We have confirmed and extended previous observations showing that the (Rh) D antigen of erythrocyte membranes is destroyed by various reagents that modify cysteine (Cys) residues (Res.) and by trypsin as well as chymotrypsin, using thirty examples of monoclonal or polyclonal anti-D in heamglutination inhibition assays. We have also shown that most C, c, E, e and BS58 epitopes are inactivated or weakened by most Cys reagents and by these proteinases, using monoclonal and polyclonal antibodies. Inactivation by 5,5-dithiobis-(2-nitrobenzoic acid) was always fully reversible after subsequent dithioerythritol treatment. The essential Cys Res. appear to be buried in the membrane in view of the inability of some reagents to inactivate (iodoacetamide, iodoacetic acid) or reactivate (reduced glutathione) the antigens. Data obtained with N-ethylmaleimide indicate that inactivation of the C and c antigens is, at least in part, attributable to (a) Cys Res. that is (are) different from that (those) involved in the E and e antigens. Data obtained with the Cys reagents and the proteinases suggest that more than one peptide loop of the Rh proteins is involved in the major Rh antigens.
Cysteine↗
Peripheral blood mononuclear phagocyte subpopulations as cellular markers in hypercholesterolemia.
Mononuclear phagocytes play a major role in the development of vascular lesions in atherogenesis. The goal of our study was to characterize circulating blood monocyte subpopulations as potential cellular markers of systemic immunological abnormalities in hypercholesterolemia. In normal subjects, three-parameter immunophenotyping of whole blood revealed that 61.3 +/- 6.0% of monocytes showed "bright" expression of the lipopolysaccharide receptor (LPSR: CD14) and Fc gamma receptor I (RI: CD64) without expression of Fc gamma-RIII (CD16). Other monocyte subsets (populations 2, 3, 4, and 5) were characterized by the simultaneous expression of both Fc gamma-R's (25.6 +/- 5.0%), isolated expression of Fc gamma-RIII (9.4 +/- 1.7%), or high expression of CD33 (3.7 +/- 1.1%) with only dim expression of CD14, respectively. The smallest subset of monocytes (population 5: 2.1 +/- 0.8%) differed from the predominant population of CD14brightCD64+CD16- monocytes by additional expression of neural cell adhesion molecule (N-CAM: CD56). In a group of hypercholesterolemic patients (n = 19), high density lipoprotein cholesterol levels were negatively correlated to the population size of CD64-CD16+ monocytes. In both healthy subjects (n = 55) and hypercholesterolemic patients, the rare apolipoprotein E3/E4 and E4/E4 phenotypes were associated with a tendency toward a larger population of CD64-CD16+ monocytes. Expression of the variant activation antigen CD45RA by peripheral blood mononuclear phagocytes showed a positive correlation to plasma levels of the atherogenic lipoproteins low density lipoprotein and lipoprotein(a). These data suggest that systemic abnormalities in mononuclear phagocyte subpopulations may play a role in the pathogenesis of atherosclerosis.
Adult↗
Effects of bunazosin and atenolol on serum lipids and apolipoproteins in a randomised trial.
The effects of bunazosin and atenolol on serum lipids and lipoproteins after 6 months of treatment were compared in this multicentric, double-blind, randomised trial. A total of 174 patients with mild to moderate essential hypertension from 15 hospitals in Germany and Poland was included in the study. Eighty-seven were treated with the alpha-receptor blocker bunazosin and the same number with the beta-blocker atenolol. Systolic and diastolic blood pressure decreased significantly in both groups, whereas only atenolol decreased pulse rate. In the bunazosin group HDL-cholesterol was significantly increased after 6 months of treatment, whereas all other analysed parameters remained unchanged. In the atenolol group total cholesterol, LDL-cholesterol, total triglycerides, apolipoprotein E, VLDL-cholesterol and VLDL-triglycerides were significantly increased after 6 months of therapy. There was a significant difference between bunazosin and atenolol for total cholesterol, HDL-cholesterol, LDL-cholesterol, VLDL-cholesterol, triglycerides, VLDL-triglycerides and apolipoprotein B levels. As a consequence, there was a significant difference in the atherogenic index of both groups. We conclude that bunazosin is favorable in the treatment of high blood pressure, because the coronary risk is not negatively influenced as shown for atenolol.
Adrenergic alpha-Antagonists↗
Lipoprotein(a)-apheresis in the secondary prevention of coronary heart disease.
Lipoprotein(a) [Lp(a)] is one important cause of atherosclerosis. It consists of one molecule of low density lipoprotein and an additional molecule of apo(a) linked to apoB-100 by a disulfide bridge. Apo(a)s are partially homologous to plasminogen, with one kringle 5 and 10-40 repeats of kringle 4. As there is no drug therapy available, we treated three patients who had suffered from at least one myocardial infarction and had Lp(a) as the only risk factor for atherosclerosis. Since October 1992, 186 immunoadsorption treatments have been carried out weekly with Sepharose-coupled anti-Lp(a)-columns. To achieve a reduction in Lp(a) from 78-250 mg/dL before apheresis to below 25 mg/dL immediately after apheresis, patient plasma volume had to be treated two to three times. Treatments lasted 3-5 h. Immediately reversible side-effects such as flushing and tachycardia during the first treatment were seen in 9% of immunoadsorptions. Non-specific protein loss remained tolerable, if one takes into account that the patients received approximately 1 L of ACDB with heparin as anticoagulant and some of the column-rinsing buffer. One patient's clinical condition and exercise test improved dramatically as did coronary angiography after 2 years. Another patient had no change after 1 year, the third patient showed subjective improvement and has not yet had repeat angiography after 1 year of treatment. We conclude that Lp(a)-apheresis may retard progression of atherosclerosis in patients with selective Lp(a) elevation. Further studies to support this hypothesis are needed.
Adult↗
Consensus protocol for the flow cytometric immunophenotyping of hematopoietic malignancies. Working Group on Flow Cytometry and Image Analysis.
Flow cytometry has become the preferred method for the lineage assignment and maturational analysis of malignant cells in acute leukemias and lymphomas. Multiparametric immunophenotyping allows the detection of aberrant antigen coexpression and the analysis of heterogeneity and clonality of malignant cells in leukemias and lymphomas. The complexity of multiparameter analysis techniques and the multitude of available monoclonal antibodies demand a standardization of protocols for the use of flow cytometry in clinical laboratories in order to achieve interlaboratory reproducibility. Therefore, the Working Group on Flow Cytometry and Image Analysis has started an initiative in order to establish a consensus protocol on the current methods of the phenotyping of hematological neoplasias as a basis for quality assurance and support for upcoming technologies such as quantitative analysis of antigen densities and automated knowledge-based analysis software. In addition to general recommendations on assay procedures and quality control specific recommendations are given for the selection of two-color reagent panels and data interpretation in an attempt to define a basis for cross-evaluation against the different currently established laboratory protocols.
Acute Disease↗
Calpastatin autoantibodies: detection, epitope mapping, and development of a specific peptide ELISA.
Autoantibodies against calpastatin (CAST) were detected in a 53-year-old female patient with a history of arthritis and thrombosis. The specificity of the autoantibodies was determined by screening expression cDNA libraries, sequence analysis of positive clones, and subsequent Western blotting against recombinant antigen. Because the Western blot lacked satisfactory reproducibility, an ELISA for anti-CAST antibodies was established. The major epitope recognized by 24 Western blot-positive sera was located within the C-terminal 27 amino acids. The ELISA was therefore based on a synthetic peptide representing these amino acids. The assay was calibrated with serial dilutions of a positive reference serum. Intraassay precision is high with a CV of approximately 4% for low- and high-titer samples. Interassay precision (CV) was 5.6-8.2% for sera with low and intermediate titers [10-60 arbitrary units (AU), where 1:100 dilution of the positive reference serum = 100 AU, 1:1000 dilution = 10 AU, etc.], which increases with higher titers ( > 60 AU). Among 205 healthy blood donors, the mean + 3 SD (after logarithmic transformation) was 30 AU; higher values were seen in 2.9% of 138 hospitalized patients. The newly developed ELISA will be a useful tool for further clinical studies on the association of anti-CAST antibodies to disease, because it permits rapid and reproducible analysis of patient sera.
Adolescent↗
Aprotinin has no effect on platelet activation and adhesion during cardiopulmonary bypass.
Aprotinin reduces blood loss following cardiopulmonary bypass operations (CPB) by the prevention of hyperfibrinolysis. Its influence on circulating platelets is uncertain. In this prospective trial we investigated activation, adhesion, and aggregation receptors on the platelet surface in 20 patients who underwent elective coronary artery bypass grafting. These patients were randomly assigned to receive either a high dose of aprotinin or placebo. Flow cytometry was performed to determine platelet activation [P-selectin, glycoprotein (GP) 53], adhesive (GP Ib), and aggregatory (GP IIb-IIIa) receptors on circulating platelets, before, during, and after CPB. Aprotinin had neither a significant effect on platelet activation nor on adhesive and aggregatory receptors. Plasma levels of D-dimers were measured before and after CPB to assess fibrinolytic activity. D-dimers following CPB and chest tube drainage were significantly less in the aprotinin group. We conclude that aprotinin reduces blood loss by its effect on fibrinolysis but has no direct influence on platelet function.
Adult↗
ACAT/CEH and ACEH/LAL: two key enzymes in hepatic cellular cholesterol homeostasis and their involvement in genetic disorders.
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Arteriosclerosis↗
A new mutation in the gene for lysosomal acid lipase leads to Wolman disease in an African kindred.
Cholesteryl ester storage disease (CESD) and Wolman disease (WD) are both autosomal recessive disorders associated with reduced activity and genetic defects of lysosomal acid lipase (LAL). The strikingly more severe course of WD is caused by genetic defects of LAL that leave no residual enzymatic activity. Mutations at the exon 8/intron 8 transition of the LAL gene have been identified in several CESD and WD patients and are responsible for the manifestation of the disease. We have determined the genetic defect in a 3-month-old boy of African origin affected by WD. No enzymatic activity of the lysosomal acid lipase was detectable in white blood cells and cultured fibroblasts. Analysis of his LAL cDNA and genomic DNA revealed that he was homozygous for a mutation at position -3 of the exon 8 splice donor site. A C-->T transition leads to a nonsense codon and to a premature termination of the LAL protein at amino acid 277. Due to this mutation, a shorter LAL mRNA species was also generated that lacked exon 8 and was deficient of the nonsense codon. As a consequence, the protein synthesis proceeded to the natural termination codon, but the enzyme generated had an internal deletion of 24 amino acids (254-277) and was also inactive. These findings, together with our previous observations when analyzing the mutations in WD and CESD patients lead to the conclusion that the more severe WD is due to mutations that absolutely abolish lysosomal acid lipase (LAL) enzyme activity and the cholesteryl ester storage disease phenotype is due to mutations that allow some residual LAL activity to be manifested.
Alternative Splicing↗
Fluorescence resonance energy transfer as a new method for the epitope-specific characterization of anti-platelet antibodies.
The detection and characterization of anti-platelet antibodies which are directed to HLA class I molecules or platelet-specific glycoproteins is of great importance in the diagnosis and treatment of thrombocytopenia. In this paper a simple and rapid flow cytometric assay for the epitope-specific characterization of anti-platelet antibodies is described using fluorescence resonance energy transfer (FRET). Patient platelets or test platelets preincubated with patient serum were analyzed for surface-bound immunoglobulins using R-phycoerythrin-conjugated polyclonal anti-human IgG antibodies (excitation 488 nm, emission 585 nm). In a second step, HLA class I structures, platelet-specific glycoproteins (gpIIb/IIIa, gpIb), and the Fc gamma receptor II were stained with murine monoclonal antibodies and Cyan 5-labelled polyclonal anti-mouse IgG antibodies (excitation 585 nm, emission 670 nm). Upon monochromatic fluorescence excitation with a 488 nm argon laser the efficiency of light transfer from R-phycoerythrin to Cyan 5 is a direct measure of the distance between the human platelet-bound antibody and the epitope detected by the murine monoclonal antibody (mab). The assay permits discrimination between human antibodies directed to different platelet-specific glycoproteins or HLA class I structures without interference from non-specific Fc gamma receptor-bound immune complexes and also between antibodies directed to different epitopes on glycoprotein heterodimers (e.g., gpIIb/IIIa).
Antibodies↗
Effects of fluvastatin on growth of porcine and human vascular smooth muscle cells in vitro.
The effects of fluvastatin, a new fully synthetic inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, on growth and cell-cycle kinetics of porcine and human vascular smooth muscle cells (SMC) were studied by growth curves and flow cytometric determination of cell-cycle distribution. Growth curves were obtained from counting after 2, 4, 7, 9, 11, and 14 days of incubation in Dulbecco's minimum essential medium and 10% fetal calf serum (FCS). For cell-cycle phase determination, cells were synchronized in G0 by 48 hours of incubation in serum-free medium, then stimulated by incubation in 10% FCS, with or without fluvastatin. There was a concentration-dependent decrease in the proliferation of human and porcine SMC when cells were incubated in the presence of fluvastatin. The reduction in the number of cells was significant with 10(-5) M and 10(-4) M fluvastatin. The G/S-phase transition of human and porcine vascular SMC was reduced to 50% of controls by 10(-4) M fluvastatin, as revealed by cell-cycle analysis. The effects of fluvastatin on growth kinetics and cell-cycle distribution could be completely reversed by the addition of 1 mM mevalonolactone, indicating that the fluvastatin effects are due to specific inhibition of HMG-CoA reductase. The addition of low density lipoprotein as a source of cholesterol failed to support SMC growth and phase transition. Addition of squalene or cholesterol to the culture medium also failed to normalize cell growth. It is concluded that nonsterol products synthesized from mevalonate are necessary for the growth of SMC.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗